1. Field of the Invention
The present invention generally relates to a liquid chromatography medium, a preparation method of the same, and a method for producing virus vaccine using the chromatography medium.
2. Description of Related Art
In recent years, development of biological preparation with various animals, insect cells, or microorganisms as hosts is significant. The productivity of biological preparation has been improved by high-volume fermentation and high-efficiency fermentation, thus the efficiency of the corresponding purification step is also required to be improved. Especially, as a purification process of cell-culture production, the production of biological preparation using a chromatography method has aroused great interest, and the chromatography medium for purification is expected to achieve high fluidity and high adsorption at a lowered cost.
In the field of production of vaccines against viral diseases, new manufacturing processes or production methods are also studied to replace previous methods. For example, in the field of manufacturer of influenza vaccines, efforts are focused on cell culture methods having the advantage of providing the product in a shorter period of time than the previous egg culture method. With such a change in the virus culture process, new purification processes are also studied. Particularly, chromatography methods have aroused great interest, which is capable of conveniently producing a preparation of high purity.
For example, in Non-patent Reference 1 (Biotechnology and Bioengineering 96 (2006), 932-944), purification of influenza virus cultured with Madin-Darby canine kidney (MDCK) cells by using anion exchange chromatography is reported. In Non-patent Reference 2 (J. Biotechnology 13 (2007), 309-317), adsorption of influenza virus from MDCK cells by chromatography utilizing the affinity property of lectin is proposed. Moreover, in Patent Reference 1 (Japanese Patent Publication No. 1999-51051), an example of industrial production method of Japanese Encephalitis virus from Vero cells by chromatography is disclosed.
In the field of virus adsorption and purification, efforts are made to attempts of using sulfated polysaccharides. For example, in the purification of influenza virus in Patent Reference 2 (Japanese Patent Publication No. 1986-47186), and the purification of Japanese Encephalitis virus in Patent Reference 3 (Japanese Patent Publication No. 1986-47185), cellulose particles sulfated with chlorosulfonic acid are used as gel for chromatography.
Such cellulose sulfate is also used as a ligand of chromatography medium in Patent Reference 4 (European Patent Application Publication No. 1808697), to concentrate influenza virus particles obtained from cell culture. In this reference, Cellufine Sulfate with sulfated ester as active group (manufactured by Chisso Corp.) is used as microporous cellulose particles having an exclusion limit molecular weight of 2000-4000 Da.
Furthermore, in Patent Reference 5 (International Publication No. 2008-125361), an example in which influenza virus particles from cell culture is purified by using a membrane formed by partially sulfating a cellulose membrane with chlorosulfonic acid is disclosed. Moreover, in Patent Reference 6 (Japanese Patent Publication No. 1997-503123), a method for preparing a gel suitable for adsorbing enveloped virus by controlling the introduced amount of sulfate group into the sulfated polysaccharide to be about 6 μmol/g is disclosed.
Furthermore, efforts are also made to develop a material suitable for adsorbing virus by supporting polysaccharide on the surface of particles. For example, in Patent Reference 7 (Japanese Patent Publication No. 1995-289891), an example in which a material suitable for adsorbing human immunodeficiency virus (HIV) is prepared by binding dextran sulfate onto porous polypropylene membrane is disclosed.
Moreover, in Patent Reference 8 (U.S. Pat. No. 6,537,793), an example of separating adenovirus by using chromatography medium is disclosed, in which the chromatography medium imparts flexible dextran arms to agarose particles for introducing ion exchange groups.
Furthermore, in Patent Reference 9 (International Publication No. 2008-039136), chromatography medium for purification of influenza virus developed by imparting sulfated dextran fibre to agarose particles is disclosed. When the medium is used, the troublesome separation of nucleic acid derived from the host cell with the influenza virus in preparation of vaccine can be improved.
Moreover, in Patent Reference 10 (Japanese Patent Publication No. 2001-190273), particles for concentrating virus prepared by binding sulfated polysaccharides insoluble in water to polystyrene particles is disclosed.
As shown in the above references, sulfated polysaccharides, especially cellulose sulfate, has high use value in the field of purification and concentration of enveloped virus such as HIV or influenza virus. For example, sulfated cellulose particles produced by introducing sulfate groups into cellulose particles by using suitable sulfating agents such as chlorosulfonic acid are sold under trade name Cellufine Sulfate by Chisso Corp. Cellufine Sulfate is useful as a chromatography medium, and in Reference 3, the adsorption of cellufine sulfate for various viruses is evaluated, and the application of cellufine sulfate in the field of purification of influenza virus are under development.
However, when direct introducing sulfate group into cellulose particles, the cellulose particles are simultaneously softened due to the hydrophilicity of the sulfate group, thus leading to the decrease of the fluidity, which is one of the important properties in chromatography. Therefore, the introduction of sulfate group has an upper limit.
Furthermore, as indicated in a comparative example in Patent Reference 5, the potential of insufficient adsorption capability exists for different strains of influenza viruses. The influenza virus has variety species based on the difference of H type, N type, and combinations thereof. Therefore, it can be expected that in production of influenza vaccine, the culture conditions of the varies is required to be different for each strain, and the purification process and conditions of vaccine are required to be individually studied for each strain. If chromatography medium with improved virus adsorption capability is used, constant level of adsorption can be ensured, thus it can be expected that the complexity of research of individual process will be correspondingly decreased.
As a result, in recent years, a new vaccine derived from cell culture is used to replace the previous egg culture. However, the vaccine manufacturers want to obtain a target of high purity with proteins and nucleic acids derived from the host cell removed. Therefore, as described above, high selectivity is required for the chromatography medium used in vaccine production.